Electrophoresis is the term used to describe the transport of charged particles, such as biological polymers, through a solvent by an electric field. Proteins and other charged particles can be characterized by their relative rate of movement in an electric field. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized preparative and analytical technique for the separation of proteins and peptides.
SDS-PAGE separates the proteins of interest from other proteins based upon an intrinsic charge-to-mass ratio and apparent molecular weight in the presence of SDS. The polyacrylamide gel acts as a molecular sieve through which proteins move according to size. The lower molecular weight proteins have a higher mobility. The proteins are treated with SDS and mercaptoethanol and behave as though they have a uniform shape and identical charge to mass ratio as they move through the gel by the force of the electric field. A molecular weight determination of the proteins can be made based on the migration of the protein through the gel relative to proteins of known molecular weight.
After protein separation by SDS-PAGE. it is often desirable to identify and recover an individual protein or proteins of interest from a complex mixture that has been resolved for further analysis or sequencing. Most commonly, techniques which rely on diffusion or elution are performed to remove proteins from the polyacrylamide gel. Electroelution is a technique whereby proteins are electrophoretically removed from the polyacrylamide gel for further processing or sequencing. In electroelution, the gel slab is soaked in a dye, such as Coomassie Brilliant Blue, which stains the protein previously separated by SDS-PAGE. The gel band containing the protein of interest is then excised and placed in an elution apparatus which applies an electric current that electrophoretically removes and isolates the proteins for further analysis.
A typical electroelution method is described by Hunkapiller et al., Methods in Enzym., 91: 227-236 (1983). Briefly, proteins are stained by Coomassie Brilliant Blue, excised, homogenized, and placed in an elution cell capped at both ends with dialysis membrane. The elution cell bridges two electrode chambers of an electrophoresis tank and a two-channel peristaltic pump recirculates the buffer solution held in the chambers. Using an elution buffer (e.g.. 0.1% SDS in 0.05 M NH.sub.4 HCO.sub.3), a constant voltage of 50V is applied for 12-16 hours which causes the negatively charged proteins to migrate from the gel toward the positive electrode. A second elution is run with a dialysis buffer (e.g., 0.02% SDS in 0.01 M NH.sub.4 HCO.sub.3) at 80 volts for 20-24 hours to remove the salt and SDS. Finally, the protein is precipitated to remove any remaining detergent or salt. The protein can then be analyzed by a number of techniques for sequence determination.
Another method of electroelution is described by Anderson et al., J. Virol., 12: 241 (1973), in which electroelution proceeds from a gel slice in a Pasteur pipet into a dialysis bag at the lower end of the pipet. In this method, protein can be lost due to adsorption onto the large surface area of the dialysis membrane, solutions transfers or through prolonged dialysis necessary to remove the SDS. Thus, this method is only efficient with large amounts of protein.
Although current electroelution methods provide effective recovery of proteins, the procedures are time consuming (generally one day) and the apparatus are only able to elute a limited number of samples simultaneously. Furthermore, the transfer of protein samples from the elution dialysis membrane to a concentrator or dialyzer in both of the above-described methods involves the risk of loss of sample proteins or polypeptides thereof.
An electroelutor device which elutes protein from gel pieces directly into a concentrator sample reservoir is described by S. Ihara et al. in "Recovery of Polypeptides from Polyacrylamide Gels by Electrophoretic Elution in a Centrifugation Concentrator", Analytical Biochemistry, 166 pg. 349-352 (1987). A commercially available concentrator is made to hold a sample tube of gel slices at an upper end and is then placed inverted (upside down) in a bath of elution buffer. Desired protein is eluted from the sample tube in an upward direction against gravity toward the membrane of the concentrator. After electrophoretic elution the concentrator and unit containing the bath of elution buffer is inverted. The concentrator now being reoriented to a right side up position is ready for centrifugal concentration or salt exchange without transfer of the eluted protein.
Although this method maximizes sample recovery by minimizing sample handling, the upside down orientation of the concentrator poses various disadvantages and difficulties. For example, the sample tube holding subject gel slices must be secured to the end of the concentrator and closed off by a dialysis membrane in order to allow the concentrator to be held upside down in the bath of elution buffer during elution. Also, the elution against gravity requires immediate inversion of the apparatus once supply of the electric field to the elution buffer is ceased. Otherwise eluted molecules fall back into the sample tube under the force of gravity, and the amount of actual sample recovery is decreased.
Accordingly, there is a need for an electroelution device which simplifies operation and minimizes sample loss.